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Abstract:

This invention provides: novel proteins, which are homologous to the
first Kunitz domain (K1) of lipoprotein-associated coagulation inhibitor
(LACI), and which are capable of inhibiting plasmin; uses of such novel
proteins in therapeutic, diagnostic, and clinical methods; and
polynucleotides that encode such novel proteins.

Claims:

1.-11. (canceled)

12. A method of treating or preventing a condition selected from the
group consisting of inappropriate fibrinolysis, inappropriate
fibrinogenolysis, excessive bleeding associated with thrombolytics,
post-operative bleeding and inappropriate angiogenesis, comprising
administering, to a human or other animal subject, an inhibitory amount
of a protein comprising a Kunitz domain having the formula:
Xaa1-Xaa2-Xaa3-Xaa4-Cys-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa-
15 -Xaa16-Xaa17-Xaa18-Xaa19-Arg-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27--
Xaa28-Xaa29-Cys-Xaa31-Xaa32-Phe-Xaa34-Xaa35-Xaa36-Gly-Cys-Xaa39-Xaa40-Xaa4-
1-Xaa42-Xaa43-Xaa44-Xaa45-
Xaa46-Xaa47-Xaa48-Xaa49-Xaa50-Cys-Xaa52-Xaa53-Xaa54-Cys-Xaa56-Xaa57-Xaa58-
, wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 may be absent;
Xaa10 is selected from Asp, Glu, Tyr and Gln; Xaa11 is selected from Thr,
Ala, Ser, Val and Asp; Xaa13 is selected from Pro, Leu and Ala; Xaa15 is
selected from Lys and Arg; Xaa16 is selected from Ala and Gly; Xaa17 is
selected from Arg, Lys and Ser; Xaa18 is selected from Phe and Ile; Xaa19
is selected from Glu, Gln, Asp, Pro, Gly, Ser and Ile; Xaa21 is selected
from Phe, Tyr and Trp; Xaa22 is selected from Tyr and Phe; Xaa23 is
selected from Tyr or Phe; Xaa31 is selected from Asp, Glu, Thr, Val, Gln
and Ala; Xaa32 is selected from Thr, Ala, Glu, Pro and Gln; Xaa34 is
selected from Val, Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala and Ser; Xaa35
is selected from Tyr and Trp; Xaa36 is selected from Gly and Ser; Xaa39
is selected from Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys and Met; Xaa40 is
selected from Gly and Ala; Xaa43 is selected from Asn and Gly; and Xaa45
is selected from Phe and Tyr.

13. The method of claim 12, wherein: Xaa10 is selected from Asp and Glu;
Xaa11 is selected from Thr, Ala and Ser; Xaa13 is Pro; Xaa15 is Arg;
Xaa16 is Ala; Xaa17 is Arg; Xaa18 is Phe; Xaa19 is selected from Glu and
Asp; Xaa21 is selected from Phe and Trp; Xaa22 is selected from Tyr and
Phe; Xaa23 is selected from Tyr or Phe; Xaa31 is selected from Asp and
Glu; Xaa32 is selected from Thr, Ala and Glu; Xaa34 is selected from Val,
Ile and Thr; Xaa35 is Tyr; Xaa36 is Gly; Xaa39 is selected from Glu, Gly
and Asp; Xaa40 is selected from Gly and Ala; Xaa43 is selected from Asn
and Gly; and Xaa45 is selected from Phe and Tyr.

wherein Xaa10 is selected from the group consisting of Asp, Glu, and Tyr;
Xaa11 is selected from the group consisting of Thr, Ala, Ser, Val, and
Asp; Xaa13 is selected from the group consisting of Pro, Leu, and Ala;
Xaa15 is selected from the group consisting of Arg and Lys; Xaa16 is
selected from the group consisting of Ala and Gly; Xaa17 is selected from
the group consisting of Arg, Lys, and Ser; Xaa18 is selected from the
group consisting of Phe and Ile; Xaa19 is selected from the group
consisting of Glu, Asp, Pro, Gly, Ser, and Ile; Xaa22 is selected from
the group consisting of Tyr and Phe; Xaa23 is selected from the group
consisting of Tyr and Phe; Xaa31 is selected from the group consisting of
Asp, Glu, Thr, Val, Gln, and Ala; Xaa32 is selected from the group
consisting of Thr, Ala, Glu, Pro, and Gln; Xaa34 is selected from the
group consisting of Val, Be, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser;
Xaa35 is selected from the group consisting of Tyr and Trp; Xaa39 is
selected from the group consisting of Glu, Gly, Asp, Arg, Ala, Gln, Leu,
Lys, and Met; Xaa40 is selected from the group consisting of Gly and Ala;
Xaa43 is selected from the group consisting of Asn and Gly; and Xaa45 is
selected from the group consisting of Phe and Tyr.

wherein Xaa10 is selected from the group consisting of Asp and Glu; Xaa11
is selected from the group consisting of Thr, Ala, Ser, Val, and Asp;
Xaa13 is selected from the group consisting of Pro, Leu, and Ala; Xaa15
is selected from the group consisting of Arg and Lys; Xaa16 is selected
from the group consisting of Ala and Gly; Xaa17 is selected from the
group consisting of Arg, Lys, and Ser; Xaa18 is selected from the group
consisting of Phe and Be; Xaa19 is selected from the group consisting of
Glu, Asp, Pro, Gly, Ser, and Ile; Xaa22 is Phe; Xaa23 is Phe; Xaa31 is
selected from the group consisting of Asp, Glu, Thr, Val, Gln, and Ala;
Xaa32 is selected from the group consisting of Thr, Ala, Glu, Pro, and
Gln; Xaa34 is selected from the group consisting of Val, Be, Thr, Leu,
Phe, Tyr, His, Asp, Ala, and Ser; Xaa35 is selected from the group
consisting of Tyr and Trp; Xaa39 is selected from the group consisting of
Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, and Met; Xaa40 is selected from
the group consisting of Gly and Ala; Xaa43 is selected from the group
consisting of Asn and Gly; and Xaa45 is selected from the group
consisting of Phe and Tyr.

16. The method of claim 12, wherein Xaa15 is Arg, Xaa16 is Ala, Xaa17 is
Arg, and Xaa18 is Phe.

26. The method of claim 12, wherein the sequence of the Kunitz domain is
identical at residues 10-21 and 31-39 and has five or fewer differences
at residues 5-9, 22-30, and 40-55 as compared to a reference sequence
selected from the group comprising SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:43, SEQ ID NO:57, SEQ ID NO:4, and SEQ ID NO:11.

29. The method of claim 12, wherein the protein comprises a Kunitz domain
having the sequence of SEQ ID NO:40.

30. The method of claim 12, wherein the protein inhibits human plasmin
with a Ki of 100 pM or less.

31. The method of claim 12, wherein the protein inhibits human plasmin
with a Ki of 300 pM or less.

Description:

[0001] The present application is a continuation-in-part of application
Ser. No. 08/208,265 (now pending) which in turn is a continuation-in-part
of Ser. No. 08/179,658, filed Jan. 11, 1994. The entirety of each of
these applications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to novel mutants of the first Kunitz domain
(K1) of the human lipoprotein-associated coagulation inhibitor LACI,
which inhibit plasmin. The invention also relates to other modified
Kunitz domains that inhibit plasmin and to other plasmin inhibitors.

[0004] 2. Description of the Background Art

[0005] The agent mainly responsible for fibrinolysis is plasmin, the
activated form of plasminogen. Many substances can activate plasminogen,
including activated Hageman factor, streptokinase, urokinase (uPA),
tissue-type plasminogen activator (tPA), and plasma kallikrein (pKA). pKA
is both an activator of the zymogen form of urokinase and a direct
plasminogen activator.

[0006] Plasmin is undetectable in normal circulating blood, but
plasminogen, the zymogen, is present at about 3 μM. An additional,
unmeasured amount of plasminogen is bound to fibrin and other components
of the extracellular matrix and cell surfaces. Normal blood contains the
physiological inhibitor of plasmin, α2-plasmin inhibitor
(α2-PI), at about 2 μM. Plasmin and α2-PI form a
1:1 complex. Matrix or cell bound-plasmin is relatively inaccessible to
inhibition by α2-PI. Thus, activation of plasmin can exceed
the neutralizing capacity of α2-PI causing a profibrinolytic
state.

[0007] Plasmin, once formed:

[0008] i. degrades fibrin clots, sometimes prematurely;

[0009] ii. digests fibrinogen (the building material of clots) impairing
hemostasis by causing formation of friable, easily lysed clots from the
degradation products, and inhibition of platelet adhesion/aggregation by
the fibrinogen degradation products;

[0012] Robbins (ROBB87) reviewed the plasminogen-plasmin system in detail.
ROBB87 and references cited therein are hereby incorporated by reference.

[0013] Fibrinolysis and Fibrinogenolysis

[0014] Inappropriate fibrinolysis and fibrinogenolysis leading to
excessive bleeding is a frequent complication of surgical procedures that
require extracorporeal circulation, such as cardiopulmonary bypass, and
is also encountered in thrombolytic therapy and organ transplantation,
particularly liver. Other clinical conditions characterized by high
incidence of bleeding diathesis include liver cirrhosis, amyloidosis,
acute promyelocytic leukemia, and solid tumors. Restoration of hemostasis
requires infusion of plasma and/or plasma products, which risks
immunological reaction and exposure to pathogens, e.g. hepatitis virus
and HIV.

[0015] Very high blood loss can resist resolution even with massive
infusion. When judged life-threatening, the hemorrhage is treated with
antifibrinolytics such as ε-amino caproic acid (See HOOV93)
(EACA), tranexamic acid, or aprotinin (NEUH89). Aprotinin is also known
as Trasylol® and as Bovine Pancreatic Trypsin Inhibitor (BPTI).
Hereinafter, aprotinin will be referred to as "BPTI". EACA and tranexamic
acid only prevent plasmin from binding fibrin by binding the kringles,
thus leaving plasmin as a free protease in plasma. BPTI is a direct
inhibitor of plasmin and is the most effective of these agents. Due to
the potential for thrombotic complications, renal toxicity and, in the
case of BPTI, immunogenicity, these agents are used with caution and
usually reserved as a "last resort" (PUTT89). All three of the
antifibrinolytic agents lack target specificity and affinity and interact
with tissues and organs through uncharacterized metabolic pathways. The
large doses required due to low affinity, side effects due to lack of
specificity and potential for immune reaction and organ/tissue toxicity
augment against use of these antifibrinolytics prophylactically to
prevent bleeding or as a routine postoperative therapy to avoid or reduce
transfusion therapy. Thus, there is a need for a safe antifibrinolytic.
The essential attributes of such an agent are:

[0016] i. Neutralization of relevant target fibrinolytic enzyme(s);

[0017] ii. High affinity binding to target enzymes to minimize dose;

[0018] iii. High specificity for target, to reduce side effects; and

[0019] iv. High degree of similarity to human protein to minimize
potential immunogenicity and organ/tissue toxicity.

[0020] All of the fibrinolytic enzymes that are candidate targets for
inhibition by an efficacious antifibrinolytic are chymotrypin-homologous
serine proteases.

[0021] Excessive Bleeding

[0022] Excessive bleeding can result from deficient coagulation activity,
elevated fibrinolytic activity, or a combination of the two conditions.
In most bleeding diatheses one must control the activity of plasmin. The
clinically beneficial effect of BPTI in reducing blood loss is thought to
result from its inhibition of plasmin (KD˜0.3 nM) or of plasma
kallikrein (KD˜100 nM) or both enzymes.

[0023] GARD93 reviews currently-used thrombolytics, saying that, although
thrombolytic agents (e.g. tPA) do open blood vessels, excessive bleeding
is a serious safety issue. Although tPA and streptokinase have short
plasma half lives, the plasmin they activate remains in the system for a
long time and, as stated, the system is potentially deficient in plasmin
inhibitors. Thus, excessive activation of plasminogen can lead to a
dangerous inability to clot and injurious or fatal hemorrhage. A potent,
highly specific plasmin inhibitor would be useful in such cases.

[0024] BPTI is a potent plasmin inhibitor; it has been found, however,
that it is sufficiently antigenic that second uses require skin testing.
Furthermore, the doses of BPTI required to control bleeding are quite
high and the mechanism of action is not clear. Some say that BPTI acts on
plasmin while others say that it acts by inhibiting plasma kallikrein.
FRAE89 reports that doses of about 840 mg of BPTI to 80 open-heart
surgery patients reduced blood loss by almost half and the mean amount
transfused was decreased by 74%. Miles Inc. has recently introduced
Trasylol in USA for reduction of bleeding in surgery (See Miles product
brochure on Trasylol, which is hereby incorporated by reference.) LOHM93
suggests that plasmin inhibitors may be useful in controlling bleeding in
surgery of the eye. SHER89 reports that BPTI may be useful in limiting
bleeding in colonic surgery.

[0025] A plasmin inhibitor that is approximately as potent as BPTI or more
potent but that is almost identical to a human protein domain offers
similar therapeutic potential but poses less potential for antigenicity.

[0026] Angiogenesis:

[0027] Plasmin is the key enzyme in angiogenesis. OREI94 reports that a 38
kDa fragment of plasmin (lacking the catalytic domain) is a potent
inhibitor of metastasis, indicating that inhibition of plasmin could be
useful in blocking metastasis of tumors (FIDL94). See also ELLI92.
ELLI92, OREI94 and FIDL94 and the references cited there are hereby
incorporated by reference.

[0028] Plasmin

[0029] Plasmin is a serine protease derived from plasminogen. The
catalytic domain of plasmin (or "CatDom") cuts peptide bonds,
particularly after arginine residues and to a lesser extent after lysines
and is highly homologous to trypsin, chymotrypsin, kallikrein, and many
other serine proteases. Most of the specificity of plasmin derives from
the kringles' binding of fibrin (LUCA83, VARA83, VARA84). On activation,
the bond between ARG561-Val562 is cut, allowing the newly free
amino terminus to form a salt bridge. The kringles remain, nevertheless,
attached to the CatDom through two disulfides (COLM87, ROBB87).

[0030] BPTI has been reported to inhibit plasmin with KD of about 300
pM (SCHN86). AUER88 reports that BPTI(R15) has Ki for plasmin
of about 13 nM, suggesting that R15 is substantially worse than
K15 for plasmin binding. SCHN86 reports that BPTI in which the
residues C14 and C38 have been converted to Alanine has Ki
for plasmin of about 4.5 nM. KIDO88 reports that APP-I has Ki for
plasmin of about 75 pM (7.5×10-11 M), the most potent
inhibitor of human plasmin reported so far. DENN94a reports, however,
that APP-I inhibits plasmin with Ki=225 nM (2.25×10-7 M).
Our second and third library were designed under the assumption that
APP-I is a potent plasmin binder. The selection process did not select
APP-I residues at most locations and the report of DENN94a explains why
this happened.

[0031] With recombinant DNA techniques, it is possible to obtain a novel
protein by expressing a mutated gene encoding a mutant of the native
protein gene. Several strategies for picking mutations are known. In one
strategy, some residues are kept constant, others are randomly mutated,
and still others are mutated in a predetermined manner. This is called
"variegation" and is defined in Ladner et al. U.S. Pat. No. 5,223,409,
which is incorporated by reference.

[0032] DENN94a and DENN94b report selections of Kunitz domains based on
APP-I for binding to the complex of Tissue Factor with Factor VIIa.
They did not use LACI-K1 as parental and did not use plasmin as a target.
The highest affinity binder they obtained had KD for their target of
about 2 nM. Our first-round selectants have affinity in this range, but
our second round selectants are about 25-fold better than this.

[0033] Proteins taken from a particular species are assumed to be less
likely to cause an immune response when injected into individuals of that
species. Murine antibodies are highly antigenic in humans. "Chimeric"
antibodies having human constant domains and murine variable domains are
decidedly less antigenic. So called "humanized" antibodies have human
constant domains and variable domains in which the CDRs arc taken from
murine antibodies while the framework of the variable domains are of
human origin. "Humanized" antibodies are much less antigenic than are
"chimeric" antibodies. In a "humanized" antibody, fifty to sixty residues
of the protein are of non-human origin. The proteins of this invention
comprise, in most cases, only about sixty amino acids and usually there
are ten or fewer differences between the engineered protein and the
parental protein. Although humans do develop antibodies even to human
proteins, such as human insulin, such antibodies tend to bind weakly and
the often do not prevent the injected protein from displaying its
intended biological function. Using a protein from the species to be
treated does not guarantee that there will be no immune response.
Nevertheless, picking a protein very close in sequence to a human protein
greatly reduces the risk of strong immune response in humans.

[0034] Kunitz domains are highly stable and can be produced efficiently in
yeast or other host organisms. At least ten human Kunitz domains have
been reported. Although APP-I was thought at one time to be a potent
plasmin inhibitor, there are, actually, no human Kunitz domains that
inhibit plasmin as well as does BPTI. Thus, it is a goal of the present
invention to provide sequences of Kunitz domain that are both potent
inhibitors of plasmin and close in sequence to human Kunitz domains.

[0035] The use of site-specific mutagenesis, whether nonrandom or random,
to obtain mutant binding proteins of improved activity is known in the
art, but success is not assured.

SUMMARY OF THE INVENTION

[0036] This invention relates to mutants of BPTI-homologous Kunitz domains
that potently inhibit human plasmin. In particular, this invention
relates to mutants of one domain of human LACI which are likely to be
non-immunogenic to humans, and which inhibit plasmin with KD,
preferably, of about 5 nM or less, more preferably of about 300 pM or
less, and most preferably about 100 pM or less. The invention also
relates to the therapeutic and diagnostic use of these novel proteins.

[0037] Plasmin-inhibiting proteins are useful for the prevention or
treatment of clinical conditions caused or exacerbated by plasmin,
including inappropriate fibrinolysis or fibrinogenolysis, excessive
bleeding associated with thrombolytics, post-operative bleeding, and
inappropriate androgenesis. Plasmin-binding mutants, whether or not
inhibitory, are useful for assaying plasmin in samples, in vitro, for
imaging areas of plasmin activity, in vivo, and for purification of
plasmin.

[0038] Preferred mutants QS4 and NS4 were selected from a library that
allowed about 50 million proteins having variability at positions 13, 16,
17, 18, 19, 31, 32, 34, and 39. These proteins have an amino-acid
sequence nearly identical to a human protein but inhibit plasmin with
Ki of about 2 nM (i.e. about 6-fold less potent than BPTI, but
100-fold better than APP-I).

[0039] An especially preferred protein, SPIT11, was selected from a
library allowing variability at positions 10, 11, 13, 15, 16, 17, 18, 19,
and 21 and has an affinity for plasmin which is less than 100 pM (i.e.
about 3-fold superior to BPTI in binding), and yet is much more similar
in sequence to LACI, a human protein, than to the BPTI, a bovine protein.
Other LACI-K1 mutants selected from this library and thought to have very
high affinity for plasmin include SPI15, SPI08, and SPI23. An additional
library allowing variation at positions 10, 11, 13, 15, 16, 17, 18, 19,
21, 31, 32, 34, 35, and 39 has been screened and a consensus sequence
(SPIcon1) found. Variants shown to be better than QS4, and thus more
preferred, include SPI51 and SPI47. Sequences that are likely to have
very high affinity for plasmin yet retain an essentially human amino-acid
sequence have been identified and include sequences SPI60, SPI59, SPI42,
SPI55, SPI56, SPI52, SPI46, SPI49, SPI53, SPI41, and SPI57. The
amino-acid sequence information that confers high affinity for the active
site of plasmin can be transferred to other Kunitz domains, particularly
to Kunitz domains of human origin; designs of several such proteins are
disclosed.

[0040] The preferred plasmin inhitors of the present invention fullfill
one or more of the following desiderata: 1) the Ki for plasmin is at
most 20 nM, preferably not more than about 5 nM, more preferably not more
than about 300 pM, and most preferably, not more than about 100 pm, 2)
the inhibitor comprises a Kunitz domain meeting the requirements shown in
Table 14 with residues number by reference to BPTI, 3) at the Kunitz
domain positions 12-21 and 32-39 one of the amino-acid types listed for
that position in Table 15, and 4) the inhibitor is more similar in
amino-acid sequence to a reference sequence selected from the group
SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, NS4, Human LACI-K2, Human
LACI-K3, Human collagen α3 KuDom, Human TFPI-2 DOMAIN 1, Human
TFPI-2 DOMAIN 2, Human TFPI-2 DOMAIN 3, HUMAN ITI-K1, Human ITI-K2, HUMAN
PROTEASE NEXIN-II, Human APP-I, DPI-1.1.1, DPI-1.1.2, DPI-1.1.3,
DPI-1.2.1, DPI-1.3.1, DPI-2.1, DPI-3.1.1, DPI-3.2.1, DPI-3.3.1,
DPI-4.1.1, DPI-4.2.1, DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, DPI-4.2.5,
DPI-5.1, DPI-5.2, DPI-6.1, DPI-6.2 than is the amino acid sequence of
said Kunitz domain to the sequence of BPTI.

[0041] Nomenclature

[0042] Herein, affinities are stated as KD
(KD(A,B)=[A][B]/[A-B]). A numerically smaller KD reflects
higher affinity. For the purposes of this invention, a "plasmin
inhibiting protein" is one that binds and inhibits plasmin with Ki
of about 20 nM or less. "Inhibition" refers to blocking the catalytic
activity of plasmin and so is measurable in vitro in assays using
chromogenic or fluorogenic substrates or in assays involving
macromolecules.

[0043] Amino-acid residues are discussed in three ways: full name of the
amino acid, standard three-letter code, and standard single-letter code.
Table use only the one-letter code. The text uses full names and
three-letter code where clarity requires.

[0044] For the purposed of this invention, "substantially homologous"
sequences are at least 51%, more preferably at least 80%, identical, over
any specified regions. Herein, sequences that are identical are
understood to be "substantially homologous". Sequences would still be
"substantially homologous" if within one region of at least 20 amino
acids they are sufficiently similar (51% or more) but outside the region
of comparison they differed totally. An insertion of one amino acid in
one sequence relative to the other counts as one mismatch. Most
preferably, no more than six residues, other than at termini, are
different. Preferably, the divergence in sequence, particularly in the
specified regions, is in the form of "conservative modifications".

[0045] "Conservative modifications" are defined as

[0046] (a) conservative substitutions of amino acids as defined in Table
9; and

[0047] (b) single or multiple insertions or deletions of amino acids at
termini, at domain boundaries, in loops, or in other segments of
relatively high mobility.

[0048] Preferably, except at termini, no more than about six amino acids
are inserted or deleted at any locus, and the modifications are outside
regions known to contain important binding sites.

[0049] Kunitz Domains

[0050] Herein, "Kunitz domain" and "KuDom" are used interchangeably to
mean a homologue of BPTI (not of the Kunitz soya-bean trypsin inhibitor).
A KuDom is a domain of a protein having at least 51 amino acids (and up
to about 61 amino acids) containing at least two, and preferably three,
disulfides. Herein, the residues of all Kunitz domains are numbered by
reference to BPTI (i.e. residues 1-58). Thus the first cysteine residue
is residue 5 and the last cysteine is 55. An amino-acid sequence shall,
for the purposed of this invention, be deemed a Kunitz domain if it can
be aligned, with three or fewer mismatches, to the sequence shown in
Table 14. An insertion or deletion of one residue shall count as one
mismatch. In Table 14, "x" matches any amino acid and "X" matches the
types listed for that position. Disulfides bonds link at least two of: 5
to 55, 14 to 38, and 30 to 51. The number of disulfides may be reduced by
one, but none of the standard cysteines shall be left unpaired. Thus, if
one cysteine is changed, then a compensating cysteine is added in a
suitable location or the matching cysteine is also replaced by a
non-cysteine (the latter being generally preferred). For example,
Drosophila funebris male accessory gland protease inhibitor has no
cysteine at position 5, but has a cysteine at position -1 (just before
position 1); presumably this forms a disulfide to CYS55. If
Cys14 and Cys38 are replaced, the requirement of Gly12,
(Gly or Ser)37, and Gly36 are dropped. From zero to many
residues, including additional domains (including other KuDoms), can be
attached to either end of a Kunitz domain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Protease inhibitors, such as Kunitz domains, function by binding
into the active site of the protease so that a peptide bond (the
"scissile bond") is: 1) not cleaved, 2) cleaved very slowly, or 3)
cleaved to no effect because the structure of the inhibitor prevents
release or separation of the cleaved segments. In Kunitz domains,
disulfide bonds act to hold the protein together even if exposed peptide
bonds are cleaved. From the residue on the amino side of the scissile
bond, and moving away from the bond, residues are conventionally called
P1, P2, P3, etc. Residues that follow the scissile bond are called P1',
P2', P3', etc. (SCHE67, SCHE68). It is generally accepted that each
serine protease has sites (comprising several residues) S1, S2, etc. that
receive the side groups and main-chain atoms of residues P1, P2, etc. of
the substrate or inhibitor and sites S1', S2', etc. that receive the side
groups and main-chain atoms of P1', P2', etc. of the substrate or
inhibitor. It is the interactions between the S sites and the P side
groups and main chain atoms that give the protease specificity with
respect to substrates and the inhibitors specificity with respect to
proteases. Because the fragment having the new amino terminus leaves the
protease first, many worker designing small molecule protease inhibitors
have concentrated on compounds that bind sites S1, S2, S3, etc.

[0052] LASK80 reviews protein protease inhibitors. Some inhibitors have
several reactive sites on one polypeptide chain, and these domains
usually have different sequences, specificities, and even topologies. It
is known that substituting amino acids in the P5 to P5' region
influences the specificity of an inhibitor. Previously, attention has
been focused on the P1 residue and those very close to it because these
can change the specificity from one enzyme class to another. LASK80
suggests that among KuDoms, inhibitors with P1=Lys or Arg inhibit
trypsin, those with P1=Tyr, Phe, Trp, Leu and Met inhibit chymotrypsin,
and those with P1=Ala or Ser are likely to inhibit elastase. Among the
Kaza1 inhibitors, LASK80 continues, inhibitors with P1=Leu or Met are
strong inhibitors of elastase, and in the Bowman-Kirk family elastase is
inhibited with P1=Ala, but not with P1=Leu. Such limited changes do not
provide inhibitors of truly high affinity (i.e. better than 1 to 10 nM).

[0053] Kunitz domains are defined above. The 3D structure (at high
resolution) of BPTI (the archetypal Kunitz domain) is known. One of the
X-ray structures is deposited in the Brookhaven Protein Data Bank as
"6PTI"]. The 3D structure of some BPTI homologues (EIGE90, HYNE90) are
known. At least seventy KuDom sequences are known. Known human homologues
include three KuDoms of LACI (WUNT88, GIRA89, NOVO89), two KuDoms of
Inter-α-Trypsin Inhibitor, APP-I (KIDO88), a KuDom from collagen,
and three KuDoms of TFPI-2 (SPRE94).

[0054] LACI

[0055] Lipoprotein-associated coagulation inhibitor (LACI) is a human
serum phosphoglycoprotein with a molecular weight of 39 kDa (amino-acid
sequence in Table 1) containing three KuDoms. We refer hereinafter to the
protein as LACI and to the Kunitz domains thereof as LACI-K1 (residues 50
to 107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The
cDNA sequence of LACI is reported in WUNT88. GIRA89 reports mutational
studies in which the P1 residues of each of the three KuDoms were
altered. LACI-K1 inhibits Factor VIIa (F VIIa) when F VIIa is
complexed to tissue factor and LACI-K2 inhibits Factor Xa. It is not
known whether LACI-K3 inhibits anything. Neither LACI nor any of the
KuDoms of LACI is a potent plasmin inhibitor.

[0056] KuDoms of this invention are substantially homologous with LACI-K1,
but differ in ways that confer strong plasmin inhibitory activity
discussed below. Other KuDoms of this invention are homologous to other
naturally-occurring KuDoms, particularly to other human KuDoms. For use
in humans, the proteins of this invention are designed to be more similar
in sequence to a human KuDom than to BPTI, to reduce the risk of causing
an immune response.

[0057] First Library of LACI-K1 and Selectants for Binding to Plasmin

[0058] Applicants have screened a first library of LACI-K1 for mutants
having high affinity for human plasmin and obtained the sequences shown
in Table 2 and Table 3. These sequences may be summarized as shown in
Table 16, where "preferred residues" are those appearing in at least one
of the 32 variants identified as binding plasmin. The preferences at
residues 13, 16, 17, 18 and 19 are strong, as shown in Table 17. Although
the range of types allowed at 31 and 32 is limited, the selection
indicates that an acidic group at 31 and a neutral group at 32 is
preferred. At residue 17, Arg was preferred; Lys, another positively
charged amino acid, was not in the library, and may be a suitable
substitute for Arg. Many amino-acid types at positions 34 and 39 are
consistent with high-affinity plasmin binding, but some types may hinder
binding.

[0059] It should be appreciated that Applicants have not sequenced all the
positive isolates of this or other libraries herein disclosed, and that
some of the possible proteins may not have been present in detectable
amounts.

[0060] Applicants have prepared one of the selected proteins, QS4, shown
in Table 2. QS4 inhibits plasmin with a Ki of about 2 nM. Although
this level of inhibition is less than that of BPTI, QS4 is a preferred
molecule for use in humans because it has less potential for
immunogenicity. Other proteins shown in Table 2 and Table 3 are very
likely to be potent inhibitors of plasmin and are likely to pose little
threat of antigenicity.

[0061] Second Library that Varies Residues 10-21

[0062] Applicants have prepared a second library of LACI-K1 derivatives
shown in Table 5 and allowing variation at residues 10, 11, 13, 15, 16,
17, 18, 19, and 21. This was screened for binding to plasmin and the
proteins shown in Table 6 were obtained.

[0063] "Consensus" in Table 6 is E10TGPCRARFERW21, where the
seven underscored residues differ from LACE-K1.Only acidic amino acids
(Glu:17 or Asp:15) were seen at position 10; Lys and Asn are not
acceptable. As Glu and Asp appeared with almost equal frequency, they
probably to contribute equally to binding. Acidic residues were not seen
at position 11. Thr was most common (11/32) with Ser appearing often
(9/32); Gly appeared 8 times. At 13, Pro was strongly preferred (24/32)
with Ala second at 5/32. At 15, Arg was strongly preferred (25/32), but a
few (7/32) isolates have Lys. Note that BPTI(R15 ) is a worse
plasmin inhibitor than is BPTI. At 16, Ala was preferred (22/32), but Gly
did appeared fairly often (10/32). At 17, Arg was most common (15/32),
with Lys coming second (9/32). At residues 17 and 18, APP-I has Met and
Ile. At 18, we allowed Ile or Phe. Only four isolates have Ile at 18 and
none of these have Met at 17. This was surprising in view of KIDO88, but
quite understandable in view of DENN94a. This collection of isolates has
a broad distribution at 19: (Glu:8, Pro:7, Asp:4, Ala:3, His:3, Gly:2,
Gln:2, Asn:1, Ser:1, and Arg:1), but acidic side groups are strongly
preferred over basic ones. At 21, the distribution was (Trp:16, Phe:14,
Leu:2, Cys:0); BPTI has Tyr at 21.

[0064] The binding of clonally pure phage that display one or another of
these proteins was compared to the binding of BPTI phage (Table 6).
Applicants have determined the Ki of protein SPI11 and found it to
be about 88 pM which is substantially superior to BPTI.

[0065] Third Library that Varies 10-21 and 31-39

[0066] Applicants used a pool of phage of the second library (varied at
residues 10, 11, 13, 15, 16, 17, 18, 19, and 21) that had been selected
twice for plasmin binding as a source of DNA into which variegation was
introduced at residues 31, 32, 34, 35, and 39 as shown in Table 7.

[0067] This library was screened for three rounds for binding to plasmin
and the isolates shown in Table 8 were obtained. The distribution of
amino-acid types is shown in Table 18 where "x" means the amino-acid type
was not allowed and "*" indicates the wild-type for LACI-K1.

[0068] These sequences gave a consensus in the 10-21 and 31-40 region of
E10TGPCRAKFDRW21 . . . E31AFVYGGCGG40(SPIcon1 in
Table 4). The ten underscored amino acids differ from LACI-K1. At eight
varied positions, a second type was quite common: Asp at 10, Ala at 11,
Glu at 19, Phe at 21, Thr at 31; Pro or Ser at 32, Leu or Ile at 34, and
Glu at 39. At position 17, the highly potent inhibitor SPI11 has R. Thus,
the sequence D10TGPCRARFDRF21 . . . E31AFIYGGCEG40
(DPI-1.1.1 in Table 4) differs from LACI-K1 by only six residues, matches
the selected sequences at the residues having strong consensus, and has
preferred substitutions at positions 10, 17, 21, 34, and 39. DPI-1.1.1 is
expected to have a very high affinity for plasmin and little potential
for immunogenicity in humans.

[0069] Preliminary testing of proteins SPI11, BPTI, SPI23, SPI51, SPI47,
QS4, SPI22, SPI54, and SPI43 for plasmin inhibitory activity placed them
in the order given. SPI11 is significantly more potent than BPTI with
Ki of about 88 pM. SPI23 and SPI51 are very similar in activity and
only slightly less potent than BPTI. SPI47 is less potent than SPI51 but
better than QS4. SPI22 is weaker than QS4. SPI54 and SPI43 are not so
potent as QS4, Ki probably >4 nM.

[0070] A KuDom that is highly homologous at residues 5-55 to any one of
the sequences SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, and NS4, as
shown in Table 4, is likely to be a potent inhibitor (KD>5 nM) of
plasmin and have a low potential for antigenicity in humans. More
preferably, to have high affinity for plasmin, a KuDom would have a
sequence that is identical at residues 10-21 and 31-39 and has five or
fewer differences at residues 5-9, 22-30, and 40-55 as compared to any of
the sequences SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, and NS4.

[0071] Using the selected sequences and the binding data of selected and
natural KuDoms, we can write a recipe for a high-affinity
plasmin-inhibiting KuDom that can be applied to other human KuDom
parentals. First, the KuDom must meet the requirements in Table 14. The
substitutions shown in Table 15 are likely to confer high-affinity
plasmin inhibitory activity on any KuDom. Thus a protein that contains a
sequence that is a KuDom, as shown in Table 14, and that contains at each
of the position 12-21 and 32-39 an amino-acid type shown in Table 15 for
that position . is likely to be a potent inhibitor of human plasmin. More
preferably, the protein would have an amino-acid type shown in Table 15
for all of the positions listed in Table 15. To reduce the potential for
immune response, one should use one or another human KuDom as parental
protein to give the sequence outside the binding region.

[0072] It is likely that a protein that comprises an amino-acid sequence
that is substantially homologous to SPI11 from residue 5 through residue
55 (as shown in Table 4) and is identical to SPI11 at positions 13-19,
31, 32, 34, and 39 will inhibit human plasmin with a Ki of 5 nM or
less. SPI11 differs from LACI-K1 at 7 positions. It is not clear that
these substitutions are equally important in fostering plasmin binding
and inhibition. There are seven molecules in which one of the substituted
positions of SPI11 is changed to the residue found in LACI-K1 (i.e.
"reverted"), 21 in which two of the residues are reverted, 35 in which
three residues are reverted, 35 in which four are reverted, 21 in which
five are reverted, and seven in which six are reverted.

[0073] It is expected that those with more residues reverted will have
less affinity for plasmin but also less potential for immunogenicity. A
person skilled in the art can pick a protein of sufficient potency and
low immunogenicity from this collection of 126. It is also possible that
substitutions in SPI11 by amino acids that differ from LACI-K1 can reduce
the immunogenicity without reducing the affinity for plasmin to a degree
that makes the protein unsuitable for use as a drug.

[0074] DESIGNED KuDom Plasmin Inhibitors

[0075] Hereinafter, "DPI" will mean a "Designed Plasmin Inhibitor" that
are KuDoms that incorporate amino-acid sequence information from the SPI
series of molecules, especially SPI11. Sequences of several DPIs and
their parental proteins are given in Table 4.

[0076] Sequences DPI-1.1.1, DPI-1.1.2, DPI-1.1.3, DPI-1.1.4, DPI-1.1.5,
and DPI-1.1.6 (in Table 4) differ from LACI-K1 by 6, 5, 5, 4, 3, and 2
amino acids respectively and represent a series in which affinity for
plasmin may decrease slowly while similarity to a human sequence
increases so as to reduce likelihood of immunogenicity. The selections
from each of the libraries show that M18F is a key substitution and that
either I17K or I17R is very important. Selections from the second and
third library indicate that Arg is strongly preferred at 15, that an acid
side group at 11 is disadvantageous to binding. The highly potent
inhibitor SPI11 differs from the consensus by having R17, as does
BPTI. DPI-1.1.1 carries the mutations D11T, K15R, I17R, M18F, K19D, and
E32A, and is likely to be highly potent as a plasmin inhibitor. DPI-1.1.2
carries D11T, K15R, I17R, M18F, and K19D, and is likely to be highly
potent. DPI-1.1.3 carries the mutations D11A, K15R, I17R, M18F, and K19D
relative to LACI-K1.DPI-1.1.3 differs from DPI-1.1.2 by having A11
instead of T11; both proteins are likely to be very potent plasmin
inhibitors. DPI-1.1.4 carries the mutations I17R, M18F, K19D, and E32A
and should be quite potent. As DPI-1.1.4 has fewer of the SPI11
mutations, it may be less potent, but is also less likely to be
immunogenic. DPI-1.1.5 carries the mutations I17R, M18F, and K19D. This
protein is likely to be a good inhibitor and is less likely to be
immunogenic. DPI-1.1.6 carries only the mutations I17R and M18F but
should inhibit plasmin.

[0077] Protein DPI-1.2.1 is based on human LACI-K2 and shown in Table 4.
The mutations P11T, I13P, Y17R, I18F, T19D, R32E, K341, and L39E are
likely to confer high affinity for plasmin. Some of these substitutions
may not be necessary; in particular, P11T and T19D may not be necessary.
Other mutations that might improve the plasmin affinity include E9A,
D10E, G16A, Y21W, Y21F, R32T, K34V, and L39G.

[0078] Protein DPI-I1.3.1 (Table 4) is based on human LACI-K3. The
mutations R11T, L13P, N17R, E18F, N19D, R31E, P32E, K341, and S36G are
intended to confer high affinity for plasmin. Some of these substitutions
may not be necessary; in particular, N19D and P32E may not be necessary.
Other changes that might improve KD include D10E, N17K, F21W and
G39E.

[0079] Protein DPI-2.1 (Table 4) is a based on the human collagen α3
KuDom. The mutations E11T, T13P, D16A, F17R, I18F, L19D, A31E, R32E, and
W34I are likely to confer high affinity for plasmin. Some of these
substitutions may not be necessary; in particular, L19D and A31E may not
be necessary. Other mutations that might improve the plasmin affinity
include K9A, D10E, D16G, K20R, R32T, W34V, and G39E.

[0080] DPI-3.1.1 (Table 4) is derived from Human TFPI-2 domain 1. The
exchanges Y11T, L17R, L18F, L19D, and R31E are likely to confer high
affinity for plasmin. The mutation L19D may not be needed. Other
mutations that might foster plasmin binding include Y21W, Y21F, Q32E,
L34I, L34V, and E39G.

[0081] DPI-3.2.1 (Table 4) is derived from Human TFPI-2 domain 2. This
parental domain contains insertions after residue 9 (one residue) and 42
(two residues). The mutations (V9SVDDQC14 replaced by
V9ETGPC14), E15R, S17K, T18F, K32T, F34V, and
(H39RNRIENR44 replaced by (E39GNRNR44) are likely to
confer affinity for plasmin. Because of the need to change the number of
amino acids, DPI-3.2.1 has a higher potential for immunogenicity than do
other modified human KuDoms.

[0082] DPI-3.3.1 (Table 4) is derived from human TFPI-2, domain 3. The
substitutions E11T, L13P, S15R, N17R, V18F, T34I, and T36G are likely to
confer high affinity for plasmin. The mutations E11T, L13P, and T34I may
not be necessary. Other mutations that might foster plasmin binding
include D10E, T19D, Y21W, and G39E.

[0083] DPI-4.1.1 (Table 4) is from human ITI-K1 by assertion of S10E,
M15R, M17K, T18F, Q34V, and M39G. The mutations M39G and Q34V may not be
necessary. Other mutations that should foster plasmin binding include:
A11T, G16A, M17R, S19D, Y21W, and Y21F.

[0084] DPI-4.2.1 (Table 4) is from human M-K2 through the mutations V10,
R11T, F17R, I18F, and P34V. The mutation P34V might not be necessary.
Other mutation that should foster plasmin binding include: V10E, Q19D,
L20R, W21F, P34I, and Q39E. DPI-4.2.2 is an especially preferred protein
as it has only three mutations: R11T, F17R, and I18F. DPI-4.2.3 is an
especially preferred protein as it has only four mutations: R11T, F17R,
I18F, and L20R. DPI-4.2.4 is an especially preferred protein as it has
only five mutations: R11T, F17R, I18F, L20R, and P34V. DPI-4.2.5 carries
the muations V10E, R11T, F17R, I18F, L20R, V31E, L32T, P34V, and Q39G and
is highly likely to inhibit plasmin very potently. Each of the proteins
DPI-4.2.1, DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, and DPI-4.2.5 is very likely
to be a highly potent inhibitor of plasmin.

[0085] Before DENN94a, it was thought that APP-I was a very potent plasmin
inhibitor. Thus, it was surprising to select proteins from a library that
was designed to allow the APP-I residues at positions 10-21 which
differed strongly from APP-I. Nevertheless, APP-I can be converted into a
potent plasmin inhibitor. DPI-5.1 is derived from human APP-I (also known
as Protease Nexin-II) by mutations M17R and I18F and is likely to be a
much better plasmin inhibitor than is APP-I itself. DPI-5.2 carries the
further mutations S19D, A31E, and F34I which may foster higher affinity
for plasmin.

[0086] DPI-6.1 is derived from the HKI B9 KuDom (NORR93) by the five
substitutions: K11T, Q15R, T16A, M17R, and M18F. DPI-6.1 is likely to be
a potent plasmin inhibitor. DPI-6.2 carries the additional mutations T19D
and A34V which should foster plasmin binding.

[0087] Although BPTI is the best naturally-occurring KuDom plasmin
inhibitors known, it could be improved. DPI-7.1 is derived from BPTI by
the mutation I18F which is likely to increase the affinity for plasmin.
DPI-7.2 carries the further mutation K15R which should increase plasmin
binding. DPI-7.3 carries the added mutation R39G. DPI-7.4 carries the
mutations Y10D, K15R, I18F, I19D, Q31E, and R39G and should have a very
high affinity for plasmin.

[0088] Modificarion of Kunitz Domains

[0089] KuDoms are quite small; if this should cause a pharmacological
problem, such as excessively quick elimination from circulation, two or
more such domains may be joined. A preferred linker is a sequence of one
or more amino acids. A preferred linker is one found between repeated
domains of a human protein, especially the linkers found in human BPTI
homologues, one of which has two domains (BALD85, ALBR83b) and another of
which has three (WUNT88).

[0090] Peptide linkers have the advantage that the entire protein may then
be expressed by recombinant DNA techniques. It is also possible to use a
nonpeptidyl linker, such as one of those commonly used to form
immunogenic conjugates. An alternative means of increasing the serum
residence of a BPTI-like KuDom is to link it to polyethyleneglycol, so
called PEGylation (DAVI79).

[0091] Ways to Improve Specificity OF SPI11 and Other Kudom Plasmin
Inhibitors:

[0092] Because we have made a large part of the surface of the KuDom SPI11
complementary to the surface of plasmin, R15 is not essential for
specific binding to plasmin. Many of the enzymes in the clotting and
fibrinolytic pathways cut preferentially after Arg or Lys. Not having a
basic residue at the P1 position may give rise to greater specificity.
The variant SPI11-R15A (shown in Table 11), having an ALA at P1, is
likely to be a good plasmin inhibitor and may have higher specificity for
plasmin relative to other proteases than does SPI11. The affinity of
SPI11-R15A for plasmin is likely to be less than the affinity of SPI11
for plasmin, but the loss of affinity for other Arg/Lys-preferring
enzymes is likely to be greater and, in many applications, specificity is
more important than affinity. Other mutants that are likely to have good
affinity and very high specificity include SPI11-R15G and
SPI11-R15N-E32A. This approach could be applied to other high-affinity
plasmin inhibitors.

[0093] Increasing the Affinity of SPI11

[0094] Variation of SPI11 as shown in Table 12 and selection of binders is
likely to produce a Kunitz domain having affinity for plasmin that is
higher than SPI11. This fourth library allows variegation of the 14-38
disulfide. The two segments of DNA shown are synthesized and used with
primers in a PCR reaction to produce ds DNA that runs from NsiI to
BstEII. The primers are identical to the 5' ends of the synthetic bits
shown and of length 21 for the first and 17 for the second. As the
variability is very high, we would endeavor to obtain between 108
and 109 transformants (the more the better).

[0095] Mode of Production

[0096] Proteins of this invention may be produced by any conventional
technique, including

[0098] b) production by recombinant DNA techniques in suitable host cells,
and

[0099] c) semisynthesis, for example, by removal of undesired sequences
from LACI-K1 and coupling of synthetic replacement sequences.

[0100] Proteins disclosed herein are preferably produced, recombinantly,
in a suitable host, such as bacteria from the genera Bacillus,
Escherichia, Salmonella, Erwinia, and yeasts from the genera Hansenula,
Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, and
Schizosaccharomyces, or cultured mammalian cells such as COS-1. The more
preferred hosts are microorganisms of the species Pichia pastoris,
Bacillus subtilis, Bacillus brevis, Saccharomyces cerevisiae, Escherichia
coli and Yarrowia lipolytica. Any promoter which is functional in the
host cell may be used to control gene expression.

[0101] Preferably the proteins are secreted and, most preferably, are
obtained from conditioned medium. Secretion is the preferred route
because proteins are more likely to fold correctly and can be produced in
conditioned medium with few contaminants. Secretion is not required.

[0102] Unless there is a specific reason to include glycogroups, we prefer
proteins designed to lack N-linked glycosylation sites to reduce
potential for antigenicity of glycogroups and so that equivalent proteins
can be expressed in a wide variety of organisms including: 1) E. coli, 2)
B. subtilis, 3) P. pastoris, 4) S. cerevisiae, and 5) mammalian cells.

[0103] Several means exist for reducing the problem of host cells
producing proteases that degrade the recombinant product; see, inter alia
BANE90 and BANE91. VAND92 reports that overexpression of the B. subtilis
signal peptidase in E. coli. leads to increased expression of a
heterologous fusion protein. ANBA88 reports that addition of PMSF (a
serine proteases inhibitor) to the culture medium improved the yield of a
fusion protein.

[0104] Other factors that may affect production of these and other
proteins disclosed here include: 1) codon usage (optimizing codons for
the host is preferred), 2) signal sequence, amino-acid sequence at
intended processing sites, presence and localization of processing,
enzymes, deletion, mutation, or inhibition of various enzymes that might
alter or degrade the engineered product and mutations that make the host
more permissive in secretion (permissive secretion hosts are preferred).

[0107] Any suitable method may be used to test the compounds of this
invention. Scatchard (Ann NY Acad Sci (1949) 51:660-669) described a
classical method of measuring and analyzing binding which is applicable
to protein binding. This method requires relatively pure protein and the
ability to distinguish bound protein from unbound.

[0108] A second appropriate method of measuring KD is to measure the
inhibitory activity against the enzyme. If the KD to be measured is
in the 1 nM to 1 μM range, this method requires chromogenic or
fluorogenic substrates and tens of micrograms to milligrams of relatively
pure inhibitor. For the proteins of this invention, having KD in the
range 5 nM to 50 pM, nanograms to micrograms of inhibitor suffice. When
using this method, the competition between the inhibitor and the enzyme
substrate can give a measured Ki that is higher than the true
Measurement reported here are not so corrected because the correction
would be very small and the any correction would reduce the Ki.
Here, we use the measured Ki as a direct measure of KD.

[0109] A third method of determining the affinity of a protein for a
second material is to have the protein displayed on a genetic package,
such as M13, and measure the ability of the protein to adhere to the
immobilized "second material". This method is highly sensitive because
the genetic packages can be amplified. We obtain at least
semiquantitative values for the binding constants by use of a pH step
gradient. Inhibitors of known affinity for the protease are used to
establish standard profiles against which other phage-displayed
inhibitors are judged. Any other suitable method of measuring protein
binding may be used.

[0110] Preferably, the proteins of this invention have a KD for
plasmin of at most about 5 nM, more preferably at most about 300 pM, and
most preferably 100 pM or less. Preferably, the binding is inhibitory so
that Ki is the same as KD. The Ki of QS4 for plasmin is
about 2 nM. The Ki of SPI11 for plasmin is about 88 pM.

[0111] Pharmaceutical Methods and Preparations

[0112] The preferred subject of this invention is a mammal. The invention
is particularly useful in the treatment of humans, but is suitable for
veternary applications too.

[0113] Herein, "protection" includes "prevention", "suppression", and
"treatment". "Prevention" involves administration of drug prior to the
induction of disease. "Suppression" involves administration of drug prior
to the clinical appearance of disease. "Treatment" involves
administration of drug after the appearance of disease.

[0114] In human and veterinary medicine, it may not be possible to
distinguish between "preventing" and "suppressing" since the inductive
event(s) may be unknown or latent, or the patient is not ascertained
until after the occurrence of the inductive event(s). We use the term
"prophylaxis" as distinct from "treatment" to encompass "preventing" and
"suppressing". Herein, "protection" includes "prophylaxis". Protection
need not by absolute to be useful.

[0115] Proteins of this invention may be administered, by any means,
systemically or topically, to protect a subject against a disease or
adverse condition. For example, administration of such a composition may
be by any parenteral route, by bolus injection or by gradual perfusion.
Alternatively, or concurrently, administration may be by the oral route.
A suitable regimen comprises administration of an effective amount of the
protein, administered as a single dose or as several doses over a period
of hours, days, months, or years.

[0116] The suitable dosage of a protein of this invention may depend on
the age, sex, health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the desired effect.
However, the most preferred dosage can be tailored to the individual
subject, as is understood and determinable by one of skill in the art,
without undue experimentation by adjustment of the dose in ways known in
the art.

[0120] Proteins of this invention may be applied in vitro to any suitable
sample that might contain plasmin to measure the plasmin present. To do
so, the assay must include a Signal Producing System (SPS) providing a
detectable signal that depends on the amount of plasmin present. The
signal may be detected visually or instrumentally. Possible signals
include production of colored, fluorescent, or luminescent products,
alteration of the characteristics of absorption or emission of radiation
by an assay component or product, and precipitation or agglutination of a
component. or product.

[0121] The component of the SPS most intimately associated with the
diagnostic reagent is called the "label". A label may be, e.g., a
radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate,
an electron-dense compound, or an agglutinable particle. A radioactive
isotope can be detected by use of, for example, a γ counter or a
scintillation counter or by autoradiography. Isotopes which are
particularly useful are 3H, 125I, 131I, 35S,
14C, and, preferably, 125I. It is also possible to label a
compound with a fluorescent compound. When the fluorescently labeled
compound is exposed to light of the proper wave length, its presence can
be detected. Among the most commonly used fluorescent labelling compounds
are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine. Alternatively,
fluorescence-emitting metals, such as 125Eu or other lanthanide, may
be attached to the binding protein using such metal chelating groups as
diethylenetriaminepentaacetic acid or ethylenediamine-tetraacetic acid.
The proteins also can be detectably labeled by coupling to a
chemiluminescent compound, such as luminol, isolumino, theromatic
acridinium ester, imidazole, acridinium salt, and oxalate ester.
Likewise, a bioluminescent compound, such as luciferin, luciferase and
aequorin, may be used to label the binding protein. The presence of a
bioluminescent protein is determined by detecting the presence of
luminescence. Enzyme labels, such as horseradish peroxidase and alkaline
phosphatase, are preferred.

[0122] There are two basic types of assays: heterogeneous and homogeneous.
In heterogeneous assays, binding of the affinity molecule to analyte does
not affect the label; thus, to determine the amount of analyte, bound
label must be separated from free label. In homogeneous assays, the
interaction does affect the activity of the label, and analyte can be
measured without separation.

[0123] In general, a plasmin-binding protein (PBP) may be used
diagnostically in the same way that an antiplasmin antibody is used.
Thus, depending on the assay format, it may be used to assay plasmin, or,
by competitive inhibition, other substances which bind plasmin.

[0124] The sample will normally be a biological fluid, such as blood,
urine, lymph, semen, milk, or cerebrospinal fluid, or a derivative
thereof, or a biological tissue, e.g., a tissue section or homogenate.
The sample could be anything. If the sample is a biological fluid or
tissue, it may be taken from a human or other mammal, vertebrate or
animal, or from a plant. The preferred sample is blood, or a fraction or
derivative thereof.

[0125] In one embodiment, the plasmin-binding protein (PBP) is
immobilized, and plasmin in the sample is allowed to compete with a known
quantity of a labeled or specifically labelable plasmin analogue. The
"plasmin analogue" is a molecule capable of competing with plasmin for
binding to the PBP, which includes plasmin itself. It may be labeled
already, or it may be labeled subsequently by specifically binding the
label to a moiety differentiating the plasmin analogue from plasmin. The
phases arc separated, and the labeled plasmin analogue in one phase is
quantified.

[0126] In a "sandwich assay", both an insolubilized plasmin-binding agent
(PBA), and a labeled PBA are employed. The plasmin analyte is captured by
the insolubilized PBA and is tagged by the labeled PBA, forming a
tertiary complex. The reagents may be added to the sample in any order.
The PBAs may be the same or different, and only one PBA need be a PBP
according to this invention (the other may be, e.g., an antibody). The
amount of labeled PBA in the tertiary complex is directly proportional to
the amount of plasmin in the sample.

[0127] The two embodiments described above are both heterogeneous assays.
A homogeneous assay requires only that the label be affected by the
binding of the PBP to plasmin. The plasmin analyte may act as its own
label if a plasmin inhibitor is used as a diagnostic reagent.

[0128] A label may be conjugated, directly or indirectly (e.g., through a
labeled anti-PBP antibody), covalently (e.g., with SPDP) or
noncovalently, to the plasmin-binding protein, to produce a diagnostic
reagent. Similarly, the plasmin binding protein may be conjugated to a
solid phase support to form a solid phase ("capture") diagnostic reagent.
Suitable supports include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, and magnetite. The carrier can be
soluble to some extent or insoluble for the purposes of this invention.
The support material may have any structure so long as the coupled
molecule is capable of binding plasmin.

[0129] In vivo Diagnosic Uses

[0130] A Kunitz domain that binds very tightly to plasmin can be used for
in vivo imaging. Diagnostic imaging of disease foci was considered one of
the largest commercial opportunities for monoclonal antibodies, but this
opportunity has not been achieved. Despite considerable effort, only two
monoclonal antibody-based imaging agents have been approved. The
disappointing results obtained with monoclonal antibodies is due in large
measure to:

[0131] i) Inadequate affinity and/or specificity;

[0132] ii) Poor penetration to target sites;

[0133] iii) Slow clearance from nontarget sites;

[0134] iv) Immunogenicity (most are murine); and

[0135] v) High production cost and poor stability.

[0136] These limitations have led most in the diagnostic imaging field to
begin to develop peptide-based imaging agents. While potentially solving
the problems of poor penetration and slow clearance, peptide-based
imaging agents are unlikely to possess adequate affinity, specificity and
in vivo stability to be useful in most applications.

[0137] Engineered proteins are uniquely suited to the requirements for an
imaging agent. in particular the extraordinary affinity and specificity
that is obtainable by engineering small, stable, human-origin protein
domains having known in vivo clearance rates and mechanisms combine to
provide earlier, more reliable results, less toxicity/side effects, lower
production and storage cost, and greater convenience of label
preparation. Indeed, it should be possible to achieve the goal of
realtime imaging with engineered protein imaging agents. Plasmin-binding
proteins, e.g. SPI11, may be useful for localizing sites of internal
hemorrhage.

[0138] Radio-labelled binding protein may be administered to the human or
animal subject. Administration is typically by injection, e.g.,
intravenous or arterial or other means of administration in a quantity
sufficient to permit subsequent dynamic and/or static imaging using
suitable radio-detecting devices. The dosage is the smallest amount
capable of providing a diagnostically effective image, and may be
determined by means conventional in the art, using known radio-imaging
agents as guides.

[0139] Typically, the imaging is carried out on the whole body of the
subject, or on that portion of the body or organ relevant to the
condition or disease under study. The radio-labelled binding protein has
accumulated. The amount of radio-labelled binding protein accumulated at
a given point in time in relevant target organs can then be quantified.

[0140] A particularly suitable radio-detecting device is a scintillation
camera, such as a y camera. The detection device in the camera senses and
records (and optional digitizes) the radioactive decay. Digitized
information can be analyzed in any suitable way, many of which are known
in the art. For example, a time-activity analysis can illustrate uptake
through clearance of the radio-labelled binding protein by the target
organs with time.

[0141] Various factors are taken into consideration in picking an
appropriate radioisotope. The isotope is picked: to allow good quality
resolution upon imaging, to be safe for diagnostic use in humans and
animals, and, preferably, to have a short half-life so as to decrease the
amount of radiation received by the body. The radioisotope used should
preferably be pharmacologically inert, and the quantities administered
should not have substantial physiological effect. The binding protein may
be radio-labelled with different isotopes of iodine, for example
123I, 125I, or 131I (see, for example, U.S. Pat. No.
4,609,725). The amount of labeling must be suitably monitored.

[0142] In applications to human subjects, it may be desirable to use
radioisotopes other than for labelling to decrease the total dosimetry
exposure of the body and to optimize the detectability of the labelled
molecule. Considering ready clinical availability for use in humans,
preferred radio-labels include: 99mTc, 67Ga, 68Ga,
90Y, 111In, .sup.113mIn, 123I, 286Re, 188Re or
211At. Radio-labelled protein may be prepared by various methods.
These include radio-halogenation by the chloramine-T or lactoperoxidase
method and subsequent purification by high pressure liquid
chromatography, for example, see Gutkowska et al in "Endocrinology and
Metabolism Clinics of America: (1987) 16 (1):183. Other methods of
radio-labelling can be used, such as IODOBEADS®.

[0143] A radio-labelled protein may be administered by any means that
enables the active agent to reach the agent's site of action in a mammal.
Because proteins are subject to digestion when administered orally,
parenteral administration, i.e., intravenous subcutaneous, intramuscular,
would ordinarily be used to optimize absorption.

[0144] Other Uses

[0145] The plasmin-binding proteins of this invention may also be used to
purify plasmin from a fluid, e.g., blood. For this purpose, the PBP is
preferably immobilized on an insoluble support. Such supports include
those already mentioned as useful in preparing solid phase diagnostic
reagents.

[0146] Proteins can be used as molecular weight markers for reference in
the separation or purification of proteins. Proteins may need to be
denatured to serve as molecular weight markers. A second general utility
for proteins is the use of hydrolyzed protein as a nutrient source.
Proteins may also be used to increase the viscosity of a solution.

[0147] The protein of this invention may be used for any of the foregoing
purposes, as well as for therapeutic and diagnostic purposes as discussed
further earlier in this specification.

[0150] As is known in the art, such methods involve blocking or protecting
reactive functional groups, such as free amino, carboxyl and thio groups.
After polypeptide bond formation, the protective groups are removed.
Thus, the addition of each amino acid residue requires several reaction
steps for protecting and deprotecting. Current methods utilize solid
phase synthesis, wherein the C-terminal amino acid is covalently linked
to an insoluble resin particles that can be filtered. Reactants are
removed by washing the resin particles with appropriate solvents using an
automated machine. Various methods, including the "tBoc" method and the
"Fmoc" method are well known in the art. See, inter alia, Atherton et
al., J Chem Soc Perkin Trans 1:538-546 (1981) and Sheppard et al., Int J
Polypeptide Prot Res 20:451-454 (1982).

EXAMPLES

Example 1

Construction of LACI (K1) Library

[0151] A synthetic oligonucleotide duplex having NsiI- and MluI-compatible
ends was cloned into a parental vector (LACI-K1::III) previously cleaved
with the above two enzymes. The resultant ligated material was
transfected by electroporation into XLIMR (F.sup.-) E. coli strain and
plated on ampicillin (Ap) plates to obtain phage-generating ApR
colonies. The variegation scheme for Phase 1 focuses on the P1 region,
and affected residues 13, 16, 17, 18 and 19. It allowed for
6.6×105 different DNA sequences (3.1×105 different
protein sequences). The library obtained consisted of 1.4×106
independent cfu's which is approximately a two fold representation of the
whole library. The phage stock generated from this plating gave a total
titer of 1.4×1013 pfu's in about 3.9 ml, with each independent
clone being represented, on average, 1×107 in total and
2.6×106 times per ml of phage stock.

[0152] To allow for variegation of residues 31, 32, 34 and 39 (phase II),
synthetic oligonucleotide duplexes with MluI- and BstEII-compatible ends
were cloned into previously cleaved Rf DNA derived from one of the
following

[0153] i) the parental construction,

[0154] ii) the phase I library, or

[0155] iii) display phage selected from the first phase binding to a given
target.

[0156] The variegation scheme for phase II allows for 4096 different DNA
sequences (1600 different protein sequences) due to alterations at
residues 31, 32, 34 and 39. The final phase II variegation is dependent
upon the level of variegation remaining following the three rounds of
binding and elution with a given target in phase I.

[0157] The combined possible variegation for both phases equals
2.7×108 different DNA sequences or 5.0×107
different protein sequences. When previously selected display phage are
used as the origin of Rf DNA for the phase II variegation, the final
level of variegation is probably in the range of 105 to 106.

[0160] i) to inoculate an F.sup.+ strain of E. coli to generate a new
display-phage stock, to be used for subsequent rounds of selection
(so-called conventional screening), or

[0161] ii) be used directly for another immediate round of selection with
the protease beads (so-called quick screening).

[0162] Typically, three rounds of either method, or a combination of the
two, are performed to give rise to the final selected display-phage from
which a representative number are sequenced and analyzed for binding
properties either as pools of display-phage or as individual clones.

[0163] For the LACI-K1 library, two phases of selection were performed,
each consisting of three rounds of binding and elution. Phase I selection
used the phase I library (variegated residues 13, 16, 17, 18, and 19)
which went through three rounds of binding and elution against plasmin
giving rise to a subpopulation of clones. The Rf DNA derived from
this selected subpopulation was used to generate the Phase II library
(addition of variegated residues 31, 32, 34 and 39). About
5.6×107 independent transformants were obtained. The phase II
libraries underwent three further rounds of binding and elution with the
same target protease giving rise to the final selectants.

[0164] Following two phases of selection against plasmin-agarose beads a
representative number (16) of final selection display-phage were
sequenced. Table 2 shows the sequences of the selected LACI-K1 domains
with the amino acids selected at the variegated positions in upper case.
Note the absolute selection of residues P13, A16, R17,
F18, and E19. There is very strong selection for E at 31 and Q
at 32. There is no consensus at 34; the observed amino acids are
{T3, Y2, H2, D, R, A, V2, I3, and L}. The amino
acids having side groups that branch at Cp (T, I, and V) are
multiply represented and are preferred. At position 39, there is no
strong consensus (G6, D3, Q2, A2, R, F, E), but G, D,
Q, and A seem to be preferred (in that order).

[0165] A separate screening of the LACI-K1 library against plasmin gave a
very similar consensus from 16 sequenced selected display-phage. These
sequences are shown in Table 3 (selected residues in upper case). These
sequences depart from those of Table 2 in that E here predominates at
position 19. There is a consensus at 34 (T5, V3, S3,
I2, L, A, F) of T, V, or S. Combining the two sets, there is a
preference for (in order of preference) T, V, I, S, A, H, Y, and L, with
F, D, and R being allowed.

[0166] Expression, Purification and Kinetic Analysis.

[0167] The three isolates QS4, ARFK#1, and ARFK#2 were recloned into a
yeast expression vector. The yeast expression vector is derived from
pMFalpha8 (KURJ82 and MIYA85). The LACI variant genes were fused to part
of the matα 1 gene, generating a hybrid gene consisting of the
matα 1 promoter-signal peptide and leader sequence-fused to the
LACI variant. The cloning site is shown in Table 24. Note that the
correctly processed LACI-K1 variant protein should be as detailed in
Table 2 and Table 3 with the addition of residues glu-ala-ala-glu to the
N-terminal met (residue 1 in Table 2 and Table 3). Expression in S.
cerevisiae gave a yield of about 500 μg of protease inhibitor per
liter of medium. Yeast-expressed LACI (kunitz domain 1), BPTI and LACI
variants: QS4, ARFK#1 and ARFK#2 were purified by affinity chromatography
using trypsin-agarose beads.

[0168] The most preferred production host is Pichia pastoris utilizing the
alcohol oxidase system. Others have produced a number of proteins in the
yeast Pichia pastoris. For example, Vedvick et al. (VEDV91) and Wagner et
al. (WAGN92) produced aprotinin from the alcohol oxidase promoter with
induction by methanol as a secreted protein in the culture medium at
≈1 mg/ml. Gregg et al. (GREG93) have reviewed production of a
number of proteins in P. pastoris. Table 1 of GREG93 shows proteins that
have been produced in P. pastoris and the yields.

[0169] Kinetic Data

[0170] Inhibition of hydrolysis of succinyl-Ala-Phe-Lys-(F3Ac)AMC (a
methyl coumarin) (Sigma Chemical, St. Louis, Mo.) by plasmin at
2.5×10-8 M with varying amount of inhibitor were fit to the
standard form for a tight-binding substrate by least-squares. Preliminary
kinetic analysis of the two AUK variants demonstrated very similar
inhibitory activity to that of the QS4 variant.) These measurements were
carried out with physiological amounts of salt (150 mM) so that the
affinities are relevant to the action of the proteins in blood.

[0171] Table 23 shows that QS4 is a highly specific inhibitor of human
plasmin. Phage that display the LACI-K1 derivative QS4 bind to plasmin
beads at least 50-times more than it binds to other protease targets.

[0172] New Library for Plasmin:

[0173] A new library of LACI-K1 domains, displayed on M13 gIIIp and
containing the diversity shown in Table 5 was made and screened for
plasmin binding. Table 6 shows the sequences selected and the consensus.
We characterized the binding of the selected proteins by comparing the
binding of clonally pure phage to BPTI display phage. Isolates 11, 15,
08, 23, and 22 were superior to BPTI phage. We produced soluble SPI11
(Selected Plasmin Inhibitor# 11) and tested its inhibitory activity,
obtaining a Ki of 88 pM which is at least two-fold better than BPTI.
Thus, we believe that the selectants SPI15, SPI08, and SPI22 are far
superior to BPTI and that SPI23 is likely to be about as potent as BPTI.
All of the listed proteins are much closer to a human protein amino-acid
sequence than is BPTI and so have less potential for immunogenicity.

[0174] All references, including those to US and foreign patents or patent
applications, and to nonpatent disclosures, are hereby incorporated by
reference in their entirety.